For purposes of this invention disclosure, the terms “lipid-soluble” and “lipophilic” refer to compounds or substances which are capable of dissolving in fats, oils, lipids, or non-polar solvents. The terms “lipid-soluble” and “lipophilic” are used interchangeably, and the term “lipophile” refers to a substance which is lipophilic.
Delivery of lipid-soluble materials such as vitamins A, D, E and K, fatty acids and lipid-soluble pharmaceuticals into the human (or animal) body remains a challenge. It can be difficult to maintain lipid-soluble nutrients in low-fat foods because they do not remain in solution and/or they adsorb to packaging materials (Swaisgood et al., 2001). Existing commercial delivery and fortification strategies revolve around emulsification and microencapsulation, both of which have limitations. Emulsification requires product-specific emulsifiers, many of which are not GRAS (Generally Recognized As Safe). Microencapsulation materials, such as cyclodextrins, are often expensive. In addition, these approaches invariably require using a substantial amount of fat as carriers for lipid-soluble materials.
The need for new carriers for lipid-soluble materials has become particularly apparent, given the recent resurgence of vitamin D deficiencies. Vitamin D is associated with bone health, myocardial development, brain and fetal development and reduced cancer risk. While the needs are evident, the means to incorporate vitamin D remain limited, at least in part due to the fact that vitamin D is sensitive to acid, oxygen, and light. Fortification of lipid-soluble vitamins, such as vitamin D, is challenging given their sensitive chemical nature. The presence of conjugated double bonds in vitamin D provides an easy route for decomposition by oxidation. Isomerization can occur under acidic or light conditions. Temperatures above 40° C. and relative humidity above 85% can deteriorate it, while mild acidification can isomerizes it to inactive forms.
Similarly, fortification of foods and beverages with fatty acids, such as polyunsaturated Ω-3 fatty acids, is very challenging because the fatty acids are highly insoluble in water and very sensitive to oxidative degradation which can reduce their health benefits and cause undesirable odors (Zimet et al., 2009).
Protein-based carriers offer a potential alternative to existing carriers, although the limited research to date on protein carriers has focused on dairy proteins. Wang et al. (1997) reported that beta-lactoglobulin, the major protein in whey, showed substantially greater binding affinity to vitamin D2 than to vitamin A. They did not, however, report being able to produce a complex using beta-lactoglobulin and a vitamin. They also did not provide binding efficiency data which would indicate what proportion of the available vitamins the protein was able to bind.
Swaisgood et al. (2001) also used beta-lactoglobulin to form a complex with vitamin D. While they were able to form a complex which was soluble in aqueous solution, their approach involved affinity purification methods, including use of affinity chromatography in their preferred method, which would be cost-prohibitive for commercial applications. The authors also did not provide information about the proportion of the added vitamin D which was retained in the complex along with beta-lactoglobulin.
Zimet et al. (2009) noted that certain food proteins, particularly milk proteins, had an ability to bind to hydrophobic molecules, making them useful for the encapsulation and delivery of bioactive compounds. They reported that beta-lactoglobulin had been found to bind with vitamin D, retinoic acid, cholesterol and various aromatic compounds and fatty acids. They noted, though, that there had been no prior published work on the binding of proteins to Ω-3 fatty acids. Using a complex containing beta-lactoglobulin and pectin, they reported an encapsulation efficiency for DHA (docosahexaenoic acid) of approximately 64% (i.e., amount of DHA encapsulated as a percent of the initially added DHA).
Semo et al. (2007) attempted to use microencapsulation involving pure casein micelles. They wrote that the use of casein micelles as carriers for nutraceuticals had not yet been reported in the literature. However, they were only able to encapsulate approximately 27% of the analytically recovered vitamin D2 which they had added to a suspension containing casein micelles.
The inventors of the present invention have unexpectedly found that using a plant-based protein, leaf protein, they were able to create a leaf protein-vitamin D complex which retained approximately 85% of the vitamin D contained in a mixture—more than three times greater percentages of vitamin D than is reported from casein micelles. The present invention pertains specifically to the use of leaf proteins in a complex with lipid-soluble materials.
The term “leaf protein” as used in this invention disclosure is intended to refer to all water-soluble proteins contained in plant leaves. The leaf protein may be obtained from any green leafy plant, as it is well known that all chlorophyll-containing plants contain soluble leaf proteins. Examples of such plants include, but are not limited to, tobacco, alfalfa and spinach. Lo et al. (2008) and Fu et al. (2010) have described a method for efficiently recovering and preparing a leaf protein powder from the leaves of green plants. Leaf protein may be extracted from plants, and a suitable leaf protein powder prepared, using the method described in Lo et al. (2008), which is incorporated by reference, or using other methods which may be known to practitioners of the art.
Leaf proteins—the proteins which occur naturally in the leaves of green plants—are perhaps the most abundant proteins in nature. They contain excellent binding, gelling, foaming, whipping and emulsifying characteristics, and have nutritional value comparable to milk protein (Lo et al., 2008; Sheen et al., 1991). Leaf protein carriers also offer another advantage over other proteins in that consumers do not have to worry about whether the products contain animal-origin or dairy-based ingredients. Leaf protein is therefore a very desirable carrier for the delivery of lipophilic substances.